PROJECT SUMMARY Patients with acute lung injury and Acute Respiratory Distress Syndrome (ARDS) have impaired gas exchange due to altered alveolar epithelial function, which results in accumulation of edema fluid leading to hypoxia. Alveolar hypoxia is common in patients with ARDS and contributes to alveolar epithelial dysfunction. Seasonal influenza infection affects a significant proportion of the population in the United States and worldwide, and while most patients infected with influenza A recover without sequelae, in some patients influenza virus infection may cause severe pneumonitis and ARDS. Alveolar epithelial cells are targets for influenza virus A, and play an important role in mounting the initial host response. Upon influenza A virus infection, alveolar epithelial cells release cytokines that contribute to the recruitment of monocytes and macrophages to the site of infection and also participate in viral clearance, which may limit the infection from spreading. However, the underlying mechanisms of these events are not completely understood. We hypothesize that in addition to its barrier function, the alveolar epithelium plays an important effector role in protecting the lung from severe injury. The first aim of this grant proposal seeks to elucidate the mechanisms that lead to the stabilization of Na,K-ATPase plasma membrane levels during hypoxia as an adaptation to stress, thus promoting cell survival. We will study whether the degradation of PKC?, which triggers the down-regulation of Na,K-ATPase, by the E3 ligase HOIL-1L decreases alveolar epithelial cell death and lung injury during chronic hypoxia. HOIL-1L is a member of the Linear Ubiquitination Assembly Complex (LUBAC). In studies proposed for the second specific aim, we will assess whether LUBAC participates in the modulation of the inflammatory intensity in the lung epithelium during influenza virus infection. The third specific aim examines the mechanisms by which increased intracellular sodium concentration, which occurs during modest inhibition of the Na,K-ATPase, prevents viral replication in alveolar epithelial cells and whether pharmacologic inhibition of the Na,K-ATPase by cardiotonic steroids such as ouabain and digoxin represents a protective mechanism by inhibiting virus replication. Understanding the mechanism(s) that lead to alveolar epithelial dysfunction caused by hypoxia and influenza virus infection will provide novel information that is of clinical relevance and has the potential for innovative approaches in the treatment of patients with acute lung injury.